Prometryn

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Dec 1987 to 21 Dec 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-3 (Estuarine/Marine Fish, Mollusk, or Shrimp Acute Toxicity Test)

Deviations:

yes

Remarks:

The protocol states that the temperature of the test solutions will be within the range of 24 - 26°C. During this study, the temperature range recorded by the continuous temperature recorder in the water bath surrounding the test vessels was 23 - 27°C.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples from each treatment level and the controls were removed and analyzed for the test substance at 0 and 96 hours. Sample for 0-hour analyses were removed from each concentration prior to the division of each solution into the replicate exposure vessels. Samples obtained at test termination were removed from one replicate solution of each treatment level and the controls. All samples were removed from the solution's mid point to avoid any undissolved material (e.g., precipitate on the bottom of the test vessel, film of undissolved material on the solution's surface).

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

A slightly cloudy stock solution of 10 mg/mL was prepared by diluting 1.02 g of the test substance with acetone to volume in a 100 mL volumetric flask. The test solutions were prepared by adding the appropriate volumes of the stock solution and dilution water in a glass flask to total 2000 mL. Each solution was then mixed with a magnetic stirrer and teflon-coated stir bar.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: Mysid shrimp
- Age at study initiation: ≤ 24 hours
- Source: The test organisms were obtained from laboratory cultures maintained at the test facility.

CULTURE CONDITIONS
- The culture water was prepared in the same manner as the dilution water used during the preliminary and definitive toxicity tests.
- Photoperiod: 16 hours/8 hours (light/ dark)
- Light intensity and location: 70 - 110 footcandles at the culture solution surfaces
- Light source: A combination of Gro-Lux and cool white fluorescent bulbs
- Feeding during cultivation: Live brine shrimp nauplii twice daily and a high protein dry food for three times weekly
- Feeding during exposure: Brine shrimp nauplii daily
- Culture temperature: 25 ± 1 °C (maintained by commercial aquarium heaters)

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

23 - 27 °C

pH:

7.9

Dissolved oxygen:

4.7 - 7.6 mg O2/L

Salinity:

32‰

Nominal and measured concentrations:

- Nominal concentrations: 0.65, 1.1, 1.8, 3.0 and 5.0 mg/L
- Measured concentrations: 0.62, 0.65, 1.8, 3.2 and 4.3 mg/L, respectively. See Table 1 in "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 3.5 cm depth x 280 cm2 surface area
- Type: closed (covered with plate glass)
- Volume of solution: 1000 mL
- Aeration: No
- No. of organisms per vessel: 10
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- No. of vessels per vehicle control: 1

TEST MEDIUM
- Source/preparation of dilution water: The dilution water was prepared by filtering natural seawater collected from the Cape Cod Canal, Bourne Massachusetts. The seawater was transferred by a pump (fiberglass reinforced thermoplastic housing) through polyvinyl chloride (PVC) pipe and transported to the laboratory in three fiberglass, 950 L holding tanks. In the laboratory, the seawater was passed through a series of polypropylene core filters (20- and 5- micron) and then recirculated within an epoxy-lined concrete reservoir prior to use. The filtered seawater was pumped to the laboratory under constant pressure through PVC pipe, an activated carbon filter and a polypropylene heat exchanger system.
- Quality control of the dilution water: Test dilution water quality was biologically monitored in the laboratory through the maintenance of continuous cultures of mysids (Mysidopsis bahia). Satisfactory survival, growth and reproduction of these sensitive organisms are used to substantiate the quality and acceptability of natural seawater used for testing marine organisms at the test facility. In conformance with EPA-GLP, routine analyses are conducted on representative samples of the seawater for the presence of pesticides and PCB's. None of these compounds have been detected in any of the water samples analyzed (detection limit 0.1 µg/L), in agreement with US EPA and ASTM standard practices.

WATER PARAMETERS
The pH, dissolved oxygen concentration, temperature and salinity were measured at 0 hour and each subsequent 24-hour exposure interval in all replicate treatment and control solutions. The temperature of the water bath was continuously monitored to maintain the exposure solution temperatures.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours/8 hours (light/dark)
- Light intensity and location: 50-90 footcandles at the surface of the solutions
- Light source: A combination of Gro-Lux and cool white fluorescent bulbs

EFFECT PARAMETERS MEASURED
Biological observations and observations of the physical characteristics of each replicate test solution were made and recorded at 0, 24, 48, 72 and 96 hours of exposure. The number of dead mysid shrimp in each replicate test solution was recorded at 24, 48, 72 and 96 hours. Dead mysids were removed daily.

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0 and 10 mg/L
- Results: The preliminary exposure of mysids to the test substance concentrations of 0.10, 1.0 and 10 mg/L resulted in mortality of 10, 30 and 100%, respectively. Based on this preliminary data the nominal concentrations selected for the definitive exposure were 0.65, 1.1, 1.8, 3.0 and 5.0 mg/L.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other:

Remarks:

95% C. I.: 1.4 - 2.0 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.65 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

The mean measured concentrations of the test substance measured in the exposure solutions at 0 and 96 hours of the exposure period were consistent between sampling intervals, averaged 89% of the nominal treatment levels, and established an exposure concentration gradient ranging from 0.62 to 4.3 mg/L.
After 48 hours of exposure to the test substance in the static system, all mysid shrimp alive in the controls and 0.65 mg/L treatment. The mean mortality of 0.62, 1.8, 3.2 and 4.3 mg/L treatments were 10, 5, 100 and 85% (one surviving mysid was lethargic), respectively. In the highest concentration (4.3 mg/L), a white, undissolved material was observed on the bottom of the test vessels, and all the surviving mysids were observed at the surface of the test solution. After 96 hours of exposure, 5% mean mortality occurred in the negative control and 0.65 mg/L treatment groups, while the mortality remained 0 in the solvent control. The mean mortality of 0.62, 1.8, 3.2 and 4.3 mg/L treatments were 10, 15, 100 and 95 %. A white, undissolved material was observed on the bottom of the highest exposure vessels. Overview of the result is provided in Table 2 in “Any other information on results incl. tables”
The 96-hour LC50 value for mysid shrimp exposed to the test substance were calculated by moving average angle analysis to be 1.7 mg/L (corresponding with 95% confidence interval of 1.4 - 2.0 mg/L). The slope of the concentration-response (mortality) curve, estimated by Probit analysis was 4.0. The NOEC for this test was 0.65 mg/L. Based on EPA criteria, the test substance would be classified as moderately toxic to Mysidopsis bahia. Overview of the result is provided in Table 3 in “Any other information on results incl. tables”

Reported statistics and error estimates:

See Statistical analyses in "Any other information on materials and methods incl. tables"

Table 2. Concentrations tested, corresponding cumulative mortalities and observations made during the 96-hour static exposure of mysid shrimp (Mysidopsis bahia) to the test substance.

Mean measured concentration (mg/L)	Cumulative mortality (%)
	24-hour			48-hour			72-hour			96-hour
	A	B	mean	A	B	mean	A	B	mean	A	B	mean
Control	0	0	0	0	0	0	0	0	0	0	10	5
Solvent control	0	0	0	0	0	0	0	0	0	0	0	0
0.62	0	0	0	10	0	10	20	0	10	20	0	10
0.65	0	0	0	0	0	0	0	0	0	10	0	5
1.8	10	0	5	10	0	5	10	10	10	20	10	15
3.2	100	80	90^c	100	100	100	100	100	100	100	100	100
4.3	80	80	80^bc	80	90	85^bcd	90	100	95^bc	90	100	95^b

a A white, undissolved material was on the test solution surface at 0-hour.
b A white undissolved test material was on the bottom of test vessels.
c All of the surviving mysids were at the surface of the test solution.
d One surviving mysid was lethargic.

Table 3.The LC50 values, 95% confidence intervals, and no observed effect concentration for mysid shrimp (Mysidopsis bahia) exposed to the test substance under static conditions.

	LC50 (mg/L)	Confidence Intervals^a
		Lower (mg/L)	Upper (mg/L)
24-hour^b	2.4	2.0	3.0
48-hour^b	2.0	1.6	2.4
72-hour^b	1.8	1.5	2.1
96-hour^b	1.7	1.4	2.0
No observed effect concentration through 96 hours was 0.65 mg/L^a.

a All calculations were based on mean measured test concentrations of the test substance.
b The LC50 value and 95% confidence interval was calculated by moving average angle analysis.

Validity criteria fulfilled:

yes

Remarks:

See the Validation criteria in the "Any other information on the materials and methods incl. tables"

Conclusions:

Based on the findings, the 96-hour NOEC was 0.65 mg/L and the LC50 value was determined to be 1.7 mg/L (corresponding 95% confidence limits of 1.4 to 2.0 mg/L).

Executive summary:

The acute toxicity of the test substance to <24-hour old mysid shrimps (Mysidopsis bahia) was studied in a 96-hour static test. The test was performed in accordance with EPA FIFRA 72-3 and in compliance with GLP. The shrimps were acclimatised before the test in natural, filtered seawater. A number of twenty mysids (10 per vessel, 2 replicate vessels per test group) were exposed to the acetone-dissolved compound at the mean measured concentrations of 0.62, 0.65, 1.8, 3.2 and 4.3 mg/L (measured by HPLC; the nominal concentrations were 0.65, 1.1, 1.8, 3.0 and 5.0 mg/L, respectively). In addition, a blank seawater and a solvent (0.5 ml acetone/L) control group were tested. The test conditions were: pH 7.6-8.0, dissolved oxygen concentration 4.4 – 7.6 mg/L and salinity 32‰. The protocol states that the temperature of the test solutions should be within the range of 24 – 26 °C. During this study, the temperature range recorded by the continuous temperature recorder in the water bath surrounding the test vessels was 23 – 27 °C. However, this deviation was not expected to have an impact on the results. All water parameters in this study were measured daily. The physical characteristics of the test solutions and the number of dead shrimps was recorded after 24, 48, 72 and 96 hours of exposure. Dead mysids were removed daily.  

 

After 48 hours of exposure to the test substance in the static system, all mysid shrimps were alive in the controls and 0.65 mg/L treatment group. The mean mortality of 0.62, 1.8, 3.2 and 4.3 mg/L treatments were 10, 5, 100 and 85% (one surviving mysid was lethargic), respectively. At the highest concentration (4.3 mg/L), a white, undissolved material was observed on the bottom of the test vessels, and all the surviving mysids were observed at the surface of the test solution. After 96 hours of exposure, 5% mean mortality occurred in the negative control and 0.65 mg/L treatment groups, while the mortality remained 0% in the solvent control. The mean mortality of the 0.62, 1.8, 3.2 and 4.3 mg/L treatments were 10, 15, 100 and 95%, respectively. A white, undissolved material was observed on the bottom of the vessel with the highest exposure concentration. Based on findings, the NOEC and LC50 values were determined to be 0.65 mg/L and 1.7 mg/L (with 95% confidence intervals of 1.4 - 2.0 mg/L), respectively. All endpoints were calculated based on the mean measured concentrations. 

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Mar 1985 to 30 Mar 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

not GLP compliant

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test was conducted according to "Standard Practice for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians" (ASTM 1980).

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

Water samples for each treatment, carrier control, and control were taken on March 28, 1985 at the time of initial test solution preparation.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

After the test concentrations were delineated via the range-finding test, a 80,000 mg/L stock solution is prepared. The stock solution was prepared by adding 2g of the test substance into 25ml acetone.
Considering the purity of the test substance (97%), the correction factor for preparing the test concentrations was calculated as 1.03. Thus, a 0.7725 mL of the stock solution was pipetted into 3 L test water to obtain the 20 mg/L solution.
Other test concentrations were obtained by diluting this 20 mg/L solution into different amount of dillution water:
- 14 mg/L test concentration: 700 mL 20 mg/L solution + 300 mL dilution water
- 10 mg/L test concentration: 500 mL 20 mg/L solution + 500 mL dilution water
- 7 mg/L test concentration: 350 mL 20 mg/L solution + 650 mL dilution water
- 5 mg/L test concentration: 250 mL 20 mg/L solution + 750 mL dilution water
Each of the five test solutions was then divided into the four replicate containers. A 500 mg/L acetone solution was used as carrier control in the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Age at study initiation: < 24 hour
- Source: Brood cultured at the test facility's bioassay laboratory

ACCLIMATION
Neonates were removed from the cultures and placed in acclimation cultures (2,000 mL beakers). The acclimation cultures were held in a water bath maintained at 20 ± 1°C. Neonates were them removed from the acclimation cultures after the adult D. Magna had released at least one prior clutch of neonates, and then were used to start tests.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

19.7 - 20.2 °C

pH:

7.65 - 7.95

Dissolved oxygen:

- 6.5 - 7.3 mg O2/L
- > 60% of saturation

Nominal and measured concentrations:

- Nominal concentrations: Control, carrier control, 5.0, 7.0, 10.0, 14.0, 20.0 mg/L
- Initial measured concentrations: 0.0, 0.0, 5.21, 6.94, 8.79, 14.82, 19.82 mg/L, respectively. See Table 1 in "Any other information on materials and methods incl. tables "

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL beakers
- Volume of solution: 80 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- No. of vessels per vehicle control: 4
- Photoperiod: 16 hours/8 hours (light/dark)
- Biomass loading rate: < 0.1g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water was used for dilutions and controls in the test.
- Hardness: 100 mg/L (as CaCO3).
- Intervals of water quality measurement: Temperature and dissolved oxygen were measured at 0 and 48 hours. The pH, conductivity, total hardness and alkalinity were measured at 0 and 48 hours in test chambers 1, 5, 9, 17 and 25.

EFFECT PARAMETERS MEASURED:
- Observation for effect: Toxicity effect criteria were recorded at 1, 4, 24, and 48 hours. Criterion of effect is immobilization of the organisms.

RANGE-FINDING STUDY
- Nominal test concentrations: control, carrier control, 2.5, 5, 10, 20 mg/L
- Study design: Two replicate test chambers, each containing 5 daphnids, were provided for each treatment level and control . The test chambers were 100 ml glass beakers containing 80 ml of test solution and dilution water. Test chambers were held in a water bath maintained at 20 ± 1°C. The 48- hour EC50 value was determined using the test substance measured concentration for each treatment. Data were analyzed by the moving average, binomial, and probit methods with the aid of a computer program following Stephen (1977)
- Results: Forty-eight hours after introduction into the test chambers, nine daphnids died and one was affected 20 mg/L, seven were affected in the 10 mg/L treatment, and no other effects were seen. Based on the binomial test, the EC50 is between 20.0 and 5.0 mg/L (confidence limit 95.0%). The moving average method produced an EC50 of 9.03 mg/L with 95% confidence limits of 7.04 and 12.24 mg/L. The probit method could not calculate an EC50.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Remarks:

Calculated based on Moving average method

Effect conc.:

12.104 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other:

Remarks:

95% C.L.: 10.998 - 13.541 mg/L

Duration:

48 h

Dose descriptor:

EC50

Remarks:

Calculated based on Probit method

Effect conc.:

12.66 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other:

Remarks:

95% C.L.:7.569 - 24.033 mg/L

Details on results:

Overview of results can be found in "Any other information on results incl. tables".
After 48 hours exposure, all daphnia were alive and mobile in the negative control group and one daphnid was dead (or immobilized) in the carrier control group. No mortality was shown in 5.21 and 6.94 mg/L treatments. While, 5 dead (or immobilized) daphnia were found in the 8.79 mg/L treatment, and this lethal number was doubled in 14.82 mg/L treatment. The highest dead (or immobilized) number of daphnia (20) was found in the highest exposure concentration (19.82 mg/L).
The Binomial test indicated that the 95% confidence limits for the EC50 was between 8.79 and 19.82 mg/L. The Moving average method produced a EC50 of 12.104 mg/L with 95% confidence limits between 10.998 and 13.541 mg/L. The Probit method determined a EC50 of 12.660 mg/L with 95% confidence limits between 7.569 and 24.003 mg/L.

The 48-hour EC50 value was determined using the measured concentration for each treatment. Data were analyzed by the moving average , binomial , and probit methods with the aid of a computer program following Stephen (1977).

Table 2 An overview of all the measured results

Measured concentration (mg/L)	Number of animals dead or immobilized at		Number of animals alive and mobile at 48 hour	Mean dissolved oxygen (mg/L)	Mean Temperature (°C)
	24 hours	48 hours
0.0 (Control)	0	0	20	7.15	20.15
0.0 (Carrier control)	0	1	19	7.25	19.95
5.21	0	0	20	6.85	20.15
6.94	0	0	20	6.90	20.15
8.79	0	5	15	6.90	20.15
14.82	1	10	10	6.90	20.15
19.82	8	20	0	6.85	20.15

Table 3 EC50 computer analysis results: Binomial test

Measured concentration (mg/L)	Number exposed	Number effected	Percent effected (%)	Binomial probability (%)
5.21	20	0	0.0	0.0001
6.94	20	0	0.0	0.0001
8.79	20	5	25.0	2.0695
14.82	20	10	50.0	58.8099
19.82	20	20	100.0	0.0001

 

Table 4 EC50 computer analysis results: Moving average method

Span	G	EC50 (mg/L)	95% confidence Limits (mg/L)
4	0.036985	12.104	10.998 - 13.541

 

Table 5 EC50 computer analysis results: Probit method

Iterations	G	H	Probability	EC50 (mg/L)	95% confidence Limits (mg/L)
7	0.696986	2.752	0.0410	12.660	7.569 – 24.003

Validity criteria fulfilled:

yes

Remarks:

See the validation criteria in "Any other information on materials and methods incl. tables "

Conclusions:

Based on the findings, the static acute toxicity test on Daphnia magna yielded 48-hour EC50 was determined to be 12.660 mg/L (with 95% confidence limits of 7.569 - 24.033 mg/L) by Probit Method and 12.104 mg/L (with 95% confidence limits of 10.998 - 13.541 mg/L) by the moving average method.

Executive summary:

The acute toxicity of the test substance to <24-hour old Daphnia magna was studied in a 48-hour static test. The test organisms were brood cultured at the test facility’s bioassay laboratory. The acclimation cultures were held in a water bath maintained at 20 ± 1 °C. Neonates were removed from the acclimation cultures after the adult daphnids had released at least one prior clutch of neonates, and then were used to start the tests. A total of twenty daphnids(5 daphnids per 100 mL glass beaker containing 80 mL test medium, 4 replicates per test group) were exposed to the acetone dissolved compound at the mean measured concentrations of 5.21, 6.94, 8.79, 14.82, 19.82 mg/L (measured by gas chromatography; the nominal concentrations were 5.0, 7.0, 10.0, 14.0, 20.0 mg/L, respectively). There were negative control and carrier control (250 ppm acetone) groups in the test as well. The test was carried out under the following conditions: temperature 19.7 - 20.2 °C, pH 7.65 - 7.95, dissolved oxygen concentration 6.5 – 7.3 mg/L (>60% of saturation). The water parameters were measured at the start and end of the test. The immobilization of the organisms was observed at 1, 4, 24 and 48 hours. The 48-hour EC50 and its 95% confidence limits were calculated by the Moving Average method with the aid of a computer program developed by US EPA.  

 

 After 48 hours of exposure, all daphnids were alive and mobile in the negative control group and one daphnia was immobilized in the carrier control group. No immobility was observed in 5.21 and 6.94 mg/L treatments. In contrast, five immobilized daphnids were found in the 8.79 mg/L treatment, and ten dead daphnids occurred in the 14.82 mg/L treatment group. All 20 daphnids were immobilized at the highest exposure concentration (19.82 mg/L). The Moving average method produced a 48-hour EC50 of 12.104 mg/L with 95% confidence limits between 10.998 and 13.541 mg/L. 

Description of key information

All available data was assessed and the studies representing the worst-case effects are included here as key. The results can be considered worst-case and are selected for the CSA.

Freshwater 48-h EC50 = 12.104 mg/L, Daphnia magna, mortality, No guideline followed; Hamaker 1985

Marine water 96-h LC50 = 1.7 mg/L, Mysidopsis bahia, mortality, EPA FIFRA TG 72 -3, Surprenant 1988

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

12.104 mg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

LC50

Effect concentration:

1.7 mg/L

Additional information

Fresh water

The acute toxicity of the test substance to <24-hour old Daphnia magna was studied in a 48-hour static test. The test organisms were brood cultured at the test facility’s bioassay laboratory. The acclimation cultures were held in a water bath maintained at 20 ± 1 °C. Neonates were removed from the acclimation cultures after the adult daphnids had released at least one prior clutch of neonates, and then were used to start the tests. A total of twenty daphnids (5 daphnids per 100 mL glass beaker containing 80 mL test medium, 4 replicates per test group) were exposed to the acetone dissolved compound at the mean measured concentrations of 5.21, 6.94, 8.79, 14.82, 19.82 mg/L (measured by gas chromatography; the nominal concentrations were 5.0, 7.0, 10.0, 14.0, 20.0 mg/L, respectively). There were negative control and carrier control (250 ppm acetone) groups in the test as well. The test was carried out under the following conditions: temperature 19.7 - 20.2 °C, pH 7.65 - 7.95, dissolved oxygen concentration 6.5 – 7.3 mg/L (>60% of saturation). The water parameters were measured at the start and end of the test. The immobilization of the organisms was observed at 1, 4, 24 and 48 hours. The 48-hour EC50 and its 95% confidence limits were calculated by the Moving Average method with the aid of a computer program developed by US EPA. After 48 hours of exposure, all daphnids were alive and mobile in the negative control group and one daphnia was immobilized in the carrier control group. No immobility was observed in 5.21 and 6.94 mg/L treatments. In contrast, five immobilized daphnids were found in the 8.79 mg/L treatment, and ten dead daphnids occurred in the 14.82 mg/L treatment group. All 20 daphnids were immobilized at the highest exposure concentration (19.82 mg/L). The Moving average method produced a 48-hour EC50 of 12.104 mg/L with 95% confidence limits between 10.998 and 13.541 mg/L. 

Marine water

The acute toxicity of the test substance to <24-hour old mysid shrimps (Mysidopsis bahia) was studied in a 96-hour static test. The test was performed in accordance with EPA FIFRA 72-3 and in compliance with GLP. The shrimps were acclimatised before the test in natural, filtered seawater. A number of twenty mysids (10 per vessel, 2 replicate vessels per test group) were exposed to the acetone-dissolved compound at the mean measured concentrations of 0.62, 0.65, 1.8, 3.2 and 4.3 mg/L (measured by HPLC; the nominal concentrations were 0.65, 1.1, 1.8, 3.0 and 5.0 mg/L, respectively). In addition, a blank seawater and a solvent (0.5 ml acetone/L) control group were tested. The test conditions were: pH 7.6-8.0, dissolved oxygen concentration 4.4 – 7.6 mg/L and salinity 32‰. The protocol states that the temperature of the test solutions should be within the range of 24 – 26 °C. During this study, the temperature range recorded by the continuous temperature recorder in the water bath surrounding the test vessels was 23 – 27 °C. However, this deviation was not expected to have an impact on the results. All water parameters in this study were measured daily. The physical characteristics of the test solutions and the number of dead shrimps was recorded after 24, 48, 72 and 96 hours of exposure. Dead mysids were removed daily. After 48 hours of exposure to the test substance in the static system, all mysid shrimps were alive in the controls and 0.65 mg/L treatment group. The mean mortality of 0.62, 1.8, 3.2 and 4.3 mg/L treatments were 10, 5, 100 and 85% (one surviving mysid was lethargic), respectively. At the highest concentration (4.3 mg/L), a white, undissolved material was observed on the bottom of the test vessels, and all the surviving mysids were observed at the surface of the test solution. After 96 hours of exposure, 5% mean mortality occurred in the negative control and 0.65 mg/L treatment groups, while the mortality remained 0% in the solvent control. The mean mortality of the 0.62, 1.8, 3.2 and 4.3 mg/L treatments were 10, 15, 100 and 95%, respectively. A white, undissolved material was observed on the bottom of the vessel with the highest exposure concentration. Based on findings, the NOEC and LC50 values were determined to be 0.65 mg/L and 1.7 mg/L (with 95% confidence intervals of 1.4 - 2.0 mg/L), respectively. All endpoints were calculated based on the mean measured concentrations.